The long term goals of this research is to analyze the molecular mechanisms involved in the adaptation of Debaryomyces hansenii to osmotic shock. To this end we have now isolated and partially characterized an osmotic shock induced promoter from this organism. Since there is some evidence that The high osmolarity glycerol (HOG) pathway is involved in this organism, we will isolate the genes involved in the pathway. We will create single deletion mutation in two genes(SSK1 and STE11 ) involved in different branches of the pathway. These mutants will then be mutagenized with conventional techniques to generate osmosensitive and glycerol constitutive phenotypes. These mutants will subsequently screened by complementation with a gene library to isolate the corresponding genes. The genes will be sequenced and compared to their counterparts in Saccharomyces. Transcription factors for the pathway will be isolated by utilzing the yeast two-hybrid system. Specific cis-acting sequences from the osmo-induced promoter will also be used to isolate proteins that bind them. These studies, in the long term will help our undestanding of environmental signal transduction in microorgansims and add to our understanding of MAP kinase pathways in yeasts. An added advantage is that it is possible, in theory, to express any gene in this organism under osmo-stress.